The recently discovered IL-33 as an IL-1 cytokine family member has been proved to be specifically released from osteonecrotic bones. We aimed to investigate the potential role of IL-33 in the development of osteonecrosis of femoral head (ONFH). Forty patients diagnosed with ONFH and forty age-, sex-, and body mass index- (BMI-) matched healthy subjects were included in this prospective study between March 2016 and September 2016. A commercially available ELISA kit was used to test the level of plasma IL-33. The IL-33 levels were compared among different ARCO stages, CJFH types, and etiology groups. Plasma IL-33 levels were significantly higher in the ONFH patients than that in the control subjects. The levels of IL-33 did not differ significantly among the ONFH patients with different ARCO stages. The IL-33 levels of patients with CJFH type L3 were significantly higher than that of patients with types L1 and L2. No significant differences were observed in IL-33 levels between steroid-induced, alcohol-induced, and idiopathic patients. Our findings seem to indicate that IL-33 effects may be detrimental during ONFH, which appeared to be associated with the prognosis of ONFH. The IL-33 deserves particular attention in the pathogenesis of ONFH. Nontraumatic osteonecrosis of the femoral head (ONFH), also known as avascular necrosis, is a refractory and progressive disease that commonly affects young patients and has a poorly understood etiology and pathogenesis . WithoutThe pathogenesis of nontraumatic ONFH is not completely clear, but it may be attributed to vascular injury, altered lipid metabolism/fat emboli, cell and bone death, mechanical stress, disruptive immune system, and defective bone repair , 6. PrevCytokines are a large group of proteins, peptides, and glycoproteins secreted by cells, which could regulate immune responses and immune response cell function by combining the corresponding receptors . CytokinThe purpose of this study was to investigate the plasma level of IL-33 in the patients with ONFH. We hypothesized that after ONFH, necrotic bone stimulates the inflammatory cytokine, IL-33, and expression and that IL-33 is an important player in the development and prognosis of ONFH. An evaluation of the role of IL-33 would reveal potential mechanisms responsible for disruptive immune responses after ONFH, which might provide a novel nonsurgical therapeutic approach for ONFH.This was a prospective clinical control study. Forty patients (40 consecutive hips) diagnosed with unilateral nontraumatic osteonecrosis of femoral head (ONFH) between March 2016 and September 2016 were included in this study. The patients were diagnosed with ONFH based on clinical history, physical examination, and radiological evaluations (X-ray and MRI) by orthopedic surgeons in our department. The inclusion criterion was a diagnosis of unilateral nontraumatic ONFH. Patients presenting the following criteria were excluded: history of trauma, active infection of the affected hip, inflammatory diseases, cardiovascular diseases, immunodeficiency, HIV infection, diabetes mellitus, renal disease, or previous surgery on the hip with ONFH. Forty age-, sex-, and body mass index- (BMI-) matched healthy subjects were simultaneously recruited as the controls. The study was approved by China-Japan Friendship Hospital (CJFH), and the methods were performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all subjects or their guardians.2 . The mean HHS for all patients was 62.6 ± 14.6 score . ONFH was idiopathic in 11 patients, secondary to steroid use in 14 patients, and associated with alcohol use in 15 patients. The patients were divided into two groups according to whether the lateral pillar of the femoral head (LPFH) was preserved [The included patients were evaluated both clinically and radiologically using the Harris hip score (HHS) , CJFH tyreserved : LPFH anThe stages by ARCO classification system were stage II in 4 patients, stage III 20 patients, and stage IV in 16 patients. All subjects with ONFH underwent an MRI or CT evaluation according to CJFH type . AccordiBlood samples were collected in sterile anticoagulation tubes from the included patients and the healthy controls, and the samples were centrifuged at 1600 rpm × 6 min to obtain plasma. The plasma was immediately frozen and stored at −80°C for analysis later. The plasma samples were tested for IL-33 levels using a commercially available ELISA kits  by following the user manual. The sensitivity of the kit is less than 2 pg/mL (range 2–500 pg/mL). The IL-33 levels were measured by two experienced independent investigators using the same instrumentation who were unaware of the study design in order to enhance measurement accuracy. All samples were duplicated during measurements. The two investigators had the same professional qualifications and were trained before the initial measurement. The data extraction and quality assessment were independently performed by two of the authors (WS. G and BL. W). If there were any disagreements, all of the authors discussed until consensus can be reached.t-tests were used to compare the IL-33 levels between different groups. One-way analysis of variance was used to compare IL-33 levels among different stage, type, and etiology groups. For statistically significant differences, groups were compared using the least significant difference (LSD) test. Pearson's correlation test was used to identify the correlation between the IL-33 levels and length of disease history. All tests were two-tailed at the 5% level of significance.The data were analyzed using SPSS version 19.0 statistical software . Quantitative variables are reported as mean ± standard deviation (SD). Nonpaired P < 0.001). The mean plasma IL-33 levels among the ONFH patients in the different etiologies, ARCO stages, and CJFH types are shown in P = 0.260).Demographic data were comparable between the two groups . Plasma P = 0.129). Although the postcollapse group showed a tendency of higher IL-33 level than the precollapse group, no significant differences were observed between the two groups (P = 0.324).The levels of plasma IL-33 were 7.54 ± 5.13 pg/mL, 14.11 ± 10.70 pg/mL, and 9.19 ± 3.38 pg/mL in the ONFH patients with ARCO stage II, stage III, and stage IV, respectively. The levels of IL-33 did not differ significantly among the ONFH patients with different ARCO stages (P = 0.003) and L2 (P = 0.028) patients, while the IL-33 levels of patients with CJFH type L2 were not significantly different with that of patients with CJFH type L1 (P = 0.210) (P = 0.034)  . Also, t= 0.034) .r = −0.136, P = 0.403).The results of Pearson's correlation test between the IL-33 levels and length of disease history for the ONFH patients showed that the IL-33 level (11.48 ± 8.34 pg/mL) did not significantly correlate with the length of disease history (38.9 ± 54.1 months) , nuclear factor of activated T cells cytoplasmic 1, c-Fos, c-Src, cathepsin K, and calcitonin receptor and eventually induced bone resorption. The property of IL-33 that regulates the inflammatory response and vascularization through modulating the recruitment and behavior of inflammatory cells may explain the indirect effect on bone remodeling during ONFH [The balance between osteoclasts and osteoblasts which determines the rate of bone remodeling could be disturbed by several molecular pathways. A variety of studies in vitro and in vivo have been conducted to investigate the effect of cytokines on osteoblasts and osteoclasts and their ultimate effect on bone resorption and formation , 24. IL-ing ONFH . In addiing ONFH , 26. As Our study showed that the ONFH patients with CJFH type L3 hips had a higher IL-33 level than those with type L1 or L2. Also, the IL-33 levels were significantly higher in the patients without LPFH than that with LPFH. Previous studies , 22 in oγ, and TNF-α [Excessive corticosteroid administration is thought to be the primary risk factor in nontraumatic osteonecrosis . Until nnd TNF-α . Interesnd TNF-α , 29, 30.nd TNF-α . In theoThe present study has some limitations. Firstly, the sample size is relatively small, and the analysis was not stratified for age and sex. Secondly, our observations were limited to inpatients, with no long-term follow-up. This marker may reflect the progression profile of osteonecrosis more precisely if it is based on the results from larger and more long-term clinical observations. Thirdly, the sensitivity and specificity of plasma IL-33 level have not been assessed. Fourth, we did not analyze the relationship between the necrotic area and the levels of plasma IL-33 in ONFH. Fifth, the controls were only matched in age, sex, and BMI, but not in other factors, such as alcohol intake. Last, the ONFH patients with stage ARCO I were not included in the study because most of them would not seek treatment in the absence of symptoms. Moreover, further studies are needed to clarify the effects of IL-33 on the natural progression outcomes and the joint-preserving surgery outcomes of patients with ONFH.In conclusion, our findings seem to indicate that IL-33 effects may be detrimental during ONFH, which appeared to be associated with the prognosis of ONFH. The application of cytokines in the prognosis and therapy of ONFH shows promise; our findings may promote the development of novel therapies for the treatment of ONFH targeting IL-33. However, further experimental and clinical studies are still needed to investigate the mechanisms of IL-33 involved in the pathogenesis of ONFH and to determine the therapeutic potential of IL-33 blockade to improve bone repair after ONFH.


The relative and absolute risks of stroke and heart failure attributable to atrial fibrillation (AF) have not been sufficiently examined.A prospective study of 23,731 community-dwelling Japanese individuals was conducted. Participants were divided into two groups based on the presence or absence of prevalent AF . Excess events (EE) due to AF and relative risks (RRs) determined using the non-AF group as the reference for incident stroke and heart failure were estimated using Poisson regression stratified by age groups  after adjustment for sex and age.There were 611 cases of stroke and 98 cases of heart failure during the observation period . AF contributed to a higher risk of stroke both in middle-aged individuals  and elderly individuals . AF also contributed to a higher risk of heart failure in middle-aged individuals  and elderly individuals . Results obtained from multivariate-adjusted analysis were similar .AF increased the risk of stroke by the same magnitude as that reported previously in Western countries. AF increased the RR of heart failure more than that in Western populations. •Incidence rates for stroke and HF were estimated in Japanese community dwellers.•Subjects were divided into AF and non-AF groups separately by age (40–69 and ≥70).•AF increased the risk of stroke by the same magnitude as in Western populations.•AF increased the risk of HF more than that in Western populations.•The absolute risk differences of outcomes were larger in the elderly individuals. The most life-threatening sequelae of atrial fibrillation (AF) are stroke and heart failure.The incidence and prevalence of AF, stroke, and heart failure all greatly increase with age.The study subjects were members of the Iwate-Kenpoku cohort (Iwate-KENCO) study. The study area consists of three local public health center-associated areas  levels, and urinary albumin:creatinine ratios (UACRs) were described previously.The initial examination consisted of a questionnaire, measurements of blood pressure and anthropometric data, blood tests, and electrocardiography (ECG). The methods used to determine serum lipid profiles, serum high-sensitivity C-reactive protein levels (hsCRP), plasma glucose levels, plasma glycosylated hemoglobin (HbA2 or higher. Hypertension (HT) was defined as systolic blood pressure (SBP) of 140 mm Hg or higher, diastolic blood pressure (DBP) of 90 mm Hg or higher, use of antihypertensive agents, or a combination thereof. Diabetes mellitus (DM) was defined as plasma glucose level of 200 mg/dL or higher, plasma HbA1c level  of 6.5% or higher, use of anti-diabetes agents, or a combination thereof among participants who provided non-fasting blood samples . In participants who provided fasting blood samples (n = 3797), subjects whose fasting glucose level was 126 mg/dL or higher were also included in the DM group, together with subjects with DM determined by the above-described definition. Dyslipidemia was defined as serum total cholesterol (TC) of 220 mg/dL or higher, serum high-density lipoprotein cholesterol (HDLC) level of less than 40 mg/dL, use of anti-hyperlipidemia agents, or a combination thereof. Regular alcohol drinking was defined as consuming alcohol on 5 days or more per week.Participants were divided into a middle-aged group (40–69 years old) and elderly group (≥70 years of age). Overweight was defined as a body mass index (BMI) of 25 kg/mThe endpoints were incident stroke, ischemic stroke, and heart failure. The methods used to ascertain the vital status of each participant were described previously.20; 8) hepatojugular reflux; 9) pulmonary edema, visceral congestion, or cardiomegaly shown on autopsy; and 10) weight loss on heart failure therapy. Minor criteria include: 1) bilateral ankle edema, 2) night cough, 3) dyspnea on ordinary exertion, 4) hepatomegaly, 5) pleural effusion by X-ray, 6) decrease in vital capacity by one-third from maximum record, 7) tachycardia (120 beats per minute or more), and 8) pulmonary vascular engorgement on chest X-ray. These criteria were checked using a registration card for hospital inpatients.The registration of heart failure was based on the criteria of the Framingham Heart Study.Continuous variables were expressed as sex- and age-adjusted means , except for hsCRP and UACR, which were expressed as sex- and age-adjusted geometric means (95% CI) using analysis of covariance (ANCOVA). The sex- and age-adjusted prevalence of each risk factor  was estimated using logistic regression analysis. Adjustment for sex was performed with a male:female ratio of 1.0, and age adjustment was performed with a 60-year-old person as the reference in the middle-aged group and a 75-year-old person as the reference in the elderly group, using ANCOVA or logistic regression.1c, logarithm-transformed hsCRP, and logarithm-transformed UACR as the references. Sex- and age-adjustment in the multivariate adjustment was performed using the same procedure as that for the sex- and age-adjusted Poisson regression analysis. All P values were two-tailed, and values less than 0.05 were considered to be statistically significant. Statistical analyses were performed using the SPSS software package, version 22  and STATA version STATA/SE 11 .We defined the follow-up period as the period from the initial survey to either the first outcome or the end of the observation period. Individuals who did not experience any outcomes in the follow-up study were administratively censored. The cumulative probability of each outcome was estimated using the Kaplan–Meier method, and differences in the cumulative probability of each outcome were tested using the log-rank test. Crude incidence rates were determined in groups stratified by sex, age, and AF status. Sex- and age-adjusted incidence rates (per 1000 person-years) and rate ratios were determined in each age group using Poisson regression analysis, which was adjusted for a male:female ratio of 1.0. Age adjustment was performed for persons aged 60 years in analysis of the middle-aged group and for persons aged 75 years in the elderly group. Excess events (incident stroke and incident heart failure) per 1000 person-years attributable to AF were determined by subtracting the adjusted incidence rate in subjects without AF from the rate in subjects with AF. Multivariate-adjusted incidence rates and rate ratios were also estimated after adjusting for explanatory variables that were statistically different between the AF and the non-AF groups in ANCOVA or logistic regression analysis. Multivariate adjustment was performed for a person with mean levels of TC, HDLC, HbAP < 0.05 using Student's t-test). The proportions of male subjects were higher in both the middle-aged and elderly individuals in the AF group (P < 0.05 using the chi-squared test). Sex- and age-adjusted mean BMI, HbA1c, hsCRP, and UACR levels were higher in the AF group than in the non-AF group in both middle-aged and elderly individuals (P < 0.05 using ANCOVA). The adjusted prevalence of overweight participants in the AF group was higher than that in the non-AF group among middle-aged individuals (P < 0.05 using logistic regression). The adjusted prevalence rate of DM was higher in middle-aged individuals in the AF group than in middle-aged individuals in the non-AF group (P < 0.05 using logistic regression).The prevalence of AF in this study was 1.4%. P < 0.01 using the log-rank test).At the completion of follow up, there were 131,088 observed person-years, and the median follow-up duration was 5.2 years. There were 611 cases of stroke and 98 cases of heart failure during the observation period. Among individuals aged less than 70 years, crude incidence rates of heart failure were 0.3 per 1000 person-years in females and 0.9 per 1000 person-years in males among individuals without AF, and the rates were 6.6 per 1000 person-years in females and 5.4 per 1000 person-years in males with AF. Among individuals aged 70 years or older, the rates were 1.7 per 1000 person-years in females and 3.2 per 1000 person-years in males without AF, and they were 12.6 per 1000 person-years in females and 24.1 per 1000 person-years in males with AF.Multivariate-adjusted analyses showed 0.6- to 1.5-times lower excess events per 1000 person-years and 0.09- to 0.96-times lower RR than those in the sex- and age-adjusted analyses. Although all excess events and relative risks attributable to AF were mildly decreased after the multivariable adjustment, similar results were obtained in the sex- and age-adjusted model and the multivariable-adjusted model.Our results showed that adjusted incidence rates and adjusted rate ratios based on Poisson regression were significantly higher for incident stroke, incident ischemic stroke, and incident heart failure in the AF group than in the non-AF group. AF increased the RRs of incident stroke, incident ischemic stroke, and incident heart failure by four to five times, seven to eight times, and seven to eight times, respectively, in the middle-aged group, and by three times, three times, and seven to eight times, respectively, in the elderly group. The absolute risk differences of outcomes expressed as excess events attributable to AF were larger in the elderly individuals than in the middle-aged individuals. Thus, AF contributed to a higher risk of stroke and heart failure in Japanese individuals, as was previously demonstrated in individuals in Western countries.9Previous studies showed that AF increased the RR of incident stroke by two to five times.The REACH registry  showed that multivariate-adjusted incidence rates of stroke were 10 per 1000 person-years in the non-AF group and 19 per 1000 person-years in the AF group.Studies on the risk of heart failure attributable to AF have not been conducted in non-Western countries. Previous studies in Western countries showed that AF increased the RR of incident heart failure by 2.35–3.4 times.The reason why the RR of heart failure due to AF was higher in our study than in previous studies might be due to the great difference between incidences of heart failure in Western and Japanese people. Among Medicare beneficiaries, age-specific incidence rates of heart failure from 1994 to 2003 were as high as 20 per 1000 person-years in persons aged 65–69 years, in the low 20 per 1000 person-years in persons aged 70–74 years, approximately 35 per 1000 person-years in persons aged 75–79 years, and approximately 50 per 1000 person-years in persons aged 80–84 years.This study has several limitations. Risk factors were only determined once, at baseline. Because AF was determined on the basis of routine examinations of 12-lead ECGs recorded by Iwate Health Service Association, some cases with paroxysmal AF were not included. Assessment of the relative risk of outcomes due to prevalent AF does not take into account the subsequent development of AF, which might have led to underestimation of the relative risk. Results based on a multivariate-adjusted model might be distorted by insufficient events per variable.The present report is the first report on the absolute risk of heart failure in individuals with AF in a Japanese general population; however, the number of incident heart failures was not sufficient for statistical analysis, especially in middle-aged individuals with or without AF and elderly individuals with AF. The estimated risk of heart failure attributable to AF obtained in our study was not thought to be robust. Further studies should be conducted to determine the risk of heart failure attributable to AF in the Japanese general population, and the RR in our study should be re-evaluated by comparison with the results of these future studies.In conclusion, adjusted rates for incident stroke, incident ischemic stroke, and incident heart failure in the AF group were higher than those in the non-AF group. AF increased the risk of stroke by the same magnitude as that determined in previous studies in Western countries. AF increased the risk of heart failure more than that in Western populations.None declared.


Escherichia coli cells, forming a functional focus. The TorR focus co-localizes with the nucleoid in a cell-cycle-dependent manner, and consequently regulates transcription of a number of genes. Formation of one TorR focus at the old poles of cells requires interaction with the MreB and DnaK proteins, and ATP, suggesting that TorR delivery requires cytoskeleton organization and ATP. Further, absence of the protein–protein interactions and ATP leads to loss in function of TorR as a transcription factor. We propose a mechanism for timing of cell-cycle-dependent gene transcription, where a transcription factor interacts with its target genes during a specific period of the cell cycle by limiting its own spatial distribution.Temporal transcriptions of genes are achieved by different mechanisms such as dynamic interaction of activator and repressor proteins with promoters, and accumulation and/or degradation of key regulators as a function of cell cycle. We find that the TorR protein localizes to the old poles of the  Caulobacter crescentus, the expression of 15%–19% of the genes varies with the cell cycle [dnaA gene (encoding the initiator for replication) and the gcrA gene . After initiation of chromosome replication, triggered by DnaA, CtrA is transcribed by stimulation of GcrA, suppressing another round of replication [Escherichia coli, an oscillatory pattern in transcription of DnaA [gidA, mioC [ftsQAZ operon [E. coli DnaA can be found in two forms: ATP-DnaA and ADP-DnaA. The ATP-DnaA is active for initiation of replication and is a strong suppressor of several genes [A complex regulatory network coordinates the progression of the cell cycle by temporal and spatial cell-cycle-dependent transcription both in bacteria and yeast ,2. In Call cycle ,4, suggelication . In Esch of DnaA , gidA, mdA, mioC  and the Z operon  is correal genes . It is hal genes . Thus, DC. crescentus [E. coli CheA/CheY and CheA/CheB two-component signal transduction systems decide the cellular chemotaxis by polar localization [Bacterial polarization is determined by dynamic changes in the subcellular localization of the signal transduction and cytoskeleton proteins as well as specific regions of the chromosome . Indeed,escentus . Similarlization . The dynlization ,16.E. coli, the TorS/TorR two-component signal transduction proteins and periplasmic TorT protein make up the trimethylamine oxide (TMAO)-regulatory system which regulates the expression of the torCAD operon encoding the TMAO-reductase anaerobic respiratory system [tor operon [tor operon [tor promoter region as does phosphorylated TorR, preventing RNA polymerase from binding to the promoter in any growth conditions [In y system . TorS ber operon . Comparer operon . Surprisnditions .lac promoter [In a screening for candidates that can be involved in control of initiation of chromosomal replication, we found that absence of TorR protein led to an early initiation of replication and the effect was indirect . Interespromoter  localizeE. coli K-12 and plasmids used in this study, are listed in RtorR:: kan allele was P1 transduced into dnaC2, ftsZ84 and minCDE mutants, respectively [ibpA gene with its native promoter region was generated by PCR using chromosomal DNA as a template and a pair of primers of ibpA-for and ibpA-rev. The mCherry gene was PCR amplified from plasmid pcDNA3-mCherry with the mix2-for and mix2-rev primers. Then, the ibpA gene with its promoter was fused to the N-terminus of the mCherry gene through a HindIII site, and the resultant ibpA-mCherry fragment was inserted into pACYC177 at XhoI and BamHI sites, constructing a plasmid, pIbpA-mCherry. For expression of the TorR-mCherry fusion protein, the torR gene with its native promoter was PCR amplified using chromosomal DNA as a template and a pair of primers of torR1-for and torR1-rev. The mCherry gene was obtained as described above. Subsequently, the torR gene with its promoter was fused to the N-terminus of mCherry gene through a KpnI site, and the resultant torR-mCherry fragment was inserted into pACYC177 at HindIII and BamHI sites, resulting in plasmid pTorR-mCherry. For expression of the MreB-mCherry fusion protein, the mreB gene with its native promoter was PCR amplified using chromosomal DNA as template and a pair of primers of mreB-for and mreB -rev. Subsequently, the mreB gene with its promoter was fused to the N-terminus of mCherry gene through a HindIII site, and the resultant mreB-mCherry fragment was inserted into pACYC177 at XhoI and BamHI sites, resulting in plasmid pMreB-mCherry. For the bacterial two-hybrid (BACTH) [torR, mreB, dnaK or ftsZ) through a proper pair of restriction enzymes as described in torR gene was amplified as described above and inserted into the pKNT25 at HindIII and BamHI sites, fusing the C-terminus of TorR to the N-terminus of the T25 domain. In the other constructions, mreB, dnaK or the ftsZ genes were PCR amplified using the primers mre-for and mre-rev for mreB, dnaK-for and dnaK-rev for dnaK, or fts-for and fts-rev for ftsZ, and inserted into pUT18 at HindIII and BamHI, fusing the C-terminus of the target protein to the N-terminus of the T18 domain. The plasmids constructed are listed in Bacterial strains which are ectively . In orde (BACTH) ,23 studiCells were exponentially grown in LB  and ABTGmCherry and 49 for Hoechst33258. Images were photographed using AxioCam MRc5 camera (Carl Zeiss) and analyzed with the Axio Vision Rel. 4.8 software (Carl Zeiss).Exponentially growing cells of the W3110 strain and its derivatives carrying the different plasmids (see text in details) in ABTGcasa or LB medium at 30 °C, 37 °C or 42 °C were collected, washed, resuspended in 1× TE buffer and fixed in 70% ethanol. Expression of the GFP fusions was induced by addition of 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) in the media. Cells were observed by Axio Imager A2 fluorescence microscopy  after 30 min of staining in Hoechst33258, which dyes DNA specifically, using chroma filter sets 13 for visualizing GFP, 43 for Total RNAs were isolated from the W3110 strain and its derivative cells using Trizol reagent  kit following the manufacturer’s instructions. RNA integrity was verified by electrophoresis on a 1.2% agarose gel containing formaldehyde, and post stained with 1.0 μg/mL ethidium bromide. The 23S/16S ratios of all samples measured using an AgilentBio analyser  were found to be about 2:1. RNA purity was determined using the NanoDrop 2000C spectrophotometer  by finding the A260/A230 and A260/A280 ratios. The A260/A280 ratios of all samples were 1.9–2.1 and the A260/A230 ratios were 2.0–2.1. Both integrity and purity of the RNA samples met the requirements for transcriptional microarray and the reverse transcriptional quantitative PCR (RT-qPCR) analysis [torRdnaC2/pTorR-GFP cells were grown to exponential phase in ABTGcasa medium with or without TorR-GFP expression at 30 °C (the permissive temperature), and then the cells were up-shifted to 42 °C (the non-permissive temperature) for 2 h to synchronize the cells. The synchronized cells were down-shifted to 30 °C and then cell samples were collected at the 0 and 45 min time points respectively  includes approximately 10,000 probe sets for all 20,366 genes present in E. coli K12 (MG1655), O157:H7-EDL933, O157:H7-Sakai and CFT073 strains and over 700 intergenic regions. For MG1655, probe sets are tiled to detect 4,358 open reading frames. The raw data was analysed by GeneChip Operating Software (Affymetrix) and the expression value for each gene of W3110∆torRdnaC2/pTorR-GFP cells at the 45 min time point was divided by the expression value at the 0 min in the presence of TorR-GFP. The same calculation between the values at the 45 min and 0 min time points was performed in the absence of TorR-GFP. Further, the two-set calculations were compared to screen the genes which could be controlled by the TorR protein in a cell-cycle-dependent manner. Two individual experiments were performed for validation. In detection analysis, we obtained “present” calls for between 3500 and 4000 genes, using target signal of 100 units. For comparison analysis, we filtered for “present” calls in both arrays. Detection and change are calculated as described in a previous report [W3110∆ectively a. The Afs report . The dats report .torRdnaC2/pTorR-GFP cells collected at 0, 15, 25, 35, 45 and 55 min after synchronization (®Premix ExTaqTMII kit (TliRNaseH Plus) (TaKaRa) was used for the RT-qPCR assay, and the reactions were performed in an Opticon 3 Real-Time PCR System  using the default cycling conditions . Melting curves were run immediately after the last cycle by reading the value at every increase of one degree for 10 s with the temperature ramping from 65 °C to 95 °C to exclude any influence of primer dimers. The Ct value for each reaction was automatically obtained, and the relative expression of target gene was calculated using the formula 2Ct), 25 mmol/L NaCl, 2 mmol/L EDTA, 0.1% Triton X-100, 1M PMSF, 10 mg/mL lysozyme and 1× protease inhibitor cocktail . The resultant lysate was shaken at 37 °C in a water bath for 30 min, frozen in liquid nitrogen and thawed by incubation at 37 °C for 15 min. The lysate was sonicated on ice for 20 min until the sample was no longer viscous. The supernatant was collected by centrifugation at 20,000× g for 15 min, and transferred to a pre-cooled 1.5 mL tube on ice. GFP-trap® beads  were equilibrated in dilution buffer containing 10 mmol/L Tris/Cl, pH7.5, 150 mmol/L NaCl, 2 mmol/L MgCl2, 1 mmol/L PMSF  and 1× protease inhibitor cocktail (Roche). The equilibrated 30 μL of bead slurry was washed in 500 μL ice-cold dilution buffer for three times at 4 °C by centrifugations and resuspensions. The cell lysate was added to the equilibrated GFP-trap® beads and incubated with gentle end-to-end rotations for 12 h at 4 °C. The beads containing proteins were collected and washed two times in 500 μL ice-cold dilution buffer. The proteins bound on beads were eluted with 2× SDS sample buffer containing 2% SDS, 20% glycerol, 2% β-mercaptoethanol, 0.5 M Tris/Cl, pH 6.8 and 0.1 mg/mL bromphenol blue dye, for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analysis.Exponentially growing cells in ABTGcasa medium at 37 °C were harvested at ODv/v) methanol, 10% (v/v) acetic acid for several hours with gentle shaking. The protein bands were cut and then analyzed by mass spectrometry. Peptides resulting from proteolytic digestion of protein gel bands were analyzed by tandem mass spectrometry using a Q Exactive LC/MS (Thermo Scientific) Liquid Chromatography-Tandem Mass Spectrometry to give a peptide mass fingerprint and additional peptide sequence information which was searched against E. coli K12-W3110 database using the Mascot search engine from Matrix Science to identify the protein as described previously [The eluted proteins were separated by SDS-PAGE, stained in Coomassie blue for 30 min with gentle shaking, following destaining in buffer containing 40%  are described above in the ‘Bacterial strains and plasmids’ section. Cells for BACTH assays were cultured at 30 °C for about 30 h on LB agar plates supplemented with ampicillin (100 μg/mL), kanamycin (50 μg/mL), 0.5 mM IPTG and chromogenic substrate X-gal (40 μg/mL).torR, pUT-mreB or pUT-dnaK plasmid . TorR-GFP localized in a focus at one cell pole  to the non-permissive temperature (42 °C)  a. New in (42 °C) . When th soluble . As show soluble b. As the soluble c. The relication . TogetheE. coli Genome GeneChip microarray assay in the presence or absence of TorR protein as a function of the cell cycle. The expression level of each gene at the 45 min relative to the 0 min time point was calculated by finding the ratio of values at 45 min to that at 0 min. The present and absent calls were validated as described previously [ftn) was up-regulated in the presence of TorR-GFP at the 45 min relative to the 0 min time point (sufABCDSE operon), iron transportation  and signal transduction (fecAR). In agreement with the findings here, expressions of fecA, fepC, ybjH, sufD were increased, and that of the ftn gene was decreased in ΔtorRΔtorSΔtorCAD cells [To address why TorR co-localizes with the nucleoid in a cell-cycle-dependent fashion, we performed an eviously . We founme point . On the me point . The resAD cells .torRdnaC2 cells containing pTorR-GFP was as described in ybjH, fhuB, fepC, fecA and sufB) out of eight tested in the presence of TorR-GFP relative to that at the 0 min gradually decreased up until 45 min and increased sharply at the 55 min time point. The fluctuations in expression of the genes accordingly responded to changes in the number of cells with the co-localized TorR and the nucleoid. The expressions reached the lowest level at the 45 min time point when TorR co-localized with the nucleoid in 73% of cells, and were increased at the 55 min time point when TorR overlapped with the nucleoid only in 36% of cells  isothiourea (A22) was used, which is a well-known inhibitor of MreB, interacting directly with MreB as a competitive inhibitor of ATP [torR cells harboring pTorR-GFP were treated with A22 for 6 h, the subcellular localization of TorR-GFP was visualized by fluorescence microscopy. Approximately 71% -dependent mode [To investigate a possible interaction of TorR with MreB or DnaK, the bacterial two-hybrid assay (BACTH)  was utilent mode , formingAlso, it was found that the fused proteins YbgI-GFP, OmpT-GFP, MalF-GFP, PstB-GFP, YcfH-GFP and OmpR-GFP localized at the cell poles, and OmpA-GFP fusions were around the outer membrane . The poltorR cells harboring pTorR-GFP were exponentially grown in LB or ABTGcasa containing IPTG with or without CCCP treatment, and the subcellular localization of TorR-GFP was investigated. We found that 77% and 41% cells had several punctae of different sizes of TorR-GFP after CCCP treatment when the cells were grown in LB and ABTGcasa medium, respectively, compared to 2%–4% in cells without CCCP treatment  to abolish the proton motive force (PMF) and subsequently the formation of ATP . W3110Δtreatment a,b. It sreatment b.ybjH, fhuB and fepC genes since these genes were shown to be regulated by TorR in a cell-cycle-dependent manner  cells harboring pTorR-GFP grown at 30 °C were shifted up to the nonpermissive temperature (42 °C) to inactivate the thermosensitive FtsZ84 protein for 2 h. Then, the distribution patterns of TorR-GFP focus or foci were investigated through fluorescence microscopy. In the absence of the MinCDE proteins, the cells were elongated and two to five TorR-GFP foci were observed along the long axis of the cells. The TorR-GFP foci were almost even in size  and the response regulator (RR) (promoter targeting) domains, respectively . To undeTo further analyze if the SR or RR domain is also needed for the protein-protein interaction, interaction of the mutant TorR with MreB or DnaK was investigated by the bacterial two-hybrid system. Deletion of the RR domain in TorR (TorR(del-RR)) abolished its interaction with MreB or DnaK . Also thfepC, fhuB and ybjH genes, in the presence of the mutant TorR-GFP proteins mentioned above, in synchronized W3110ΔtorRdnaC2 cells. RT-qPCR analysis showed that the expression levels of the fepC, fhuB and ybjH genes were significantly increased in the SR- or RR-deleted TorR or multi-mutations in TorR  [C. crescentus) [Timing of gene expression during the cell cycle, of some key genes specifically, is crucial for progression of the cell cycle and is a universal process. About 400–800 genes in eukaryotic cells including es pombe  as well es pombe  are exprscentus) ,73; (ii)scentus) ; and (iiscentus) ,75. HereE. coli cells are different [tor boxes in vivo to regulate expression of the tor operon [Distribution patterns of the response regulators of the two-component signal transduction systems in the ifferent . Severalifferent . OmpR-GFifferent . Very inifferent . We belir operon , and thi


Partial tooth agenesis is frequently observed in Robin sequence. Tooth anomalies are increasingly considered as an extended phenotype of the cleft palate population. The study objective was to compare the prevalence and patterns of tooth agenesis in a group of patients with non-syndromic Robin sequence (ns-RS) and a group with non-syndromic cleft palate (ns-CP).The panoramic radiographs of 115 ns-RS and 191 ns-CP patients were assessed for agenesis of the permanent dentition (excluding third molars) and the patterns recorded using the Tooth Agenesis Code.Partial tooth agenesis was observed in 47.8% of ns-RS and 29.8% of ns-CP patients with a greater prevalence in the mandibula than in the maxilla, particularly in ns-RS. The teeth most frequently absent in both groups were the mandibular second premolars and maxillary lateral incisors. Tooth agenesis was bilateral in two-thirds of affected ns-RS patients and one-half of ns-CP patients. In ns-RS, bilateral agenesis of the mandibular second premolars was more frequently observed in female than that in male patients. Completely symmetrical patterns of hypodontia were found in around 45% of ns-RS patients with tooth agenesis compared to 35% in ns-CP. No association was found between the extent of the palatal cleft and the severity of hypodontia.Tooth agenesis is more prevalent in ns-RS than that in ns-CP, demonstrates a much greater predilection for the mandible in ns-RS, and bears no relation to the extent of the palatal cleft.When compared to ns-CP, additional developmental disturbances are likely involved in the etiology of tooth agenesis in ns-RS. Future research could help identify the underlying genetic traits and aid in classifying patients in those with and without expected tooth agenesis in order to facilitate orthodontic management strategies. Although tooth agenesis is the most common developmental anomaly of the human permanent dentition, its etiology still remains poorly understood. Dental agenesis may occur either as an isolated trait or as part of a recognized congenital syndrome. Partial tooth agenesis or hypodontia is frequently observed in Robin sequence (RS). This congenital disorder bears the name of the French stomatologist Pierre Robin and consists of the triad of micro- or retrognathia, glossoptosis, and obstructive respiratory distress , 2. In aIn the general, population tooth agenesis (excluding third molars) is observed in 3.2 to 7.6% of all individuals . RecentlThe objective of the present study was to test the aforementioned hypothesis indirectly by determining the prevalence of tooth agenesis (excluding third molars) and its patterns in a sample of Dutch patients with ns-RS and in a comparison group of Dutch patients with ns-CP. In order to record the pattern of tooth agenesis per individual patient, the study employed the Tooth Agenesis Code (TAC) which is a method that attaches a unique value to each pattern of agenesis , 12. As NVSCA) in order to facilitate epidemiological and clinical research  , 12, whiA total of 115 patients with ns-RS were included in the study group and 191 patients with ns-CP in the comparison group, see Table Table In ns-RS, a total of 139 teeth were absent with a median of 2 per patient and a range of one to ten teeth per patient. Table N = 11/55) of ns-RS patients with tooth agenesis and was observed more frequently in female patients . The most prevalent patterns with agenesis of the lateral incisors were bilateral mandibular/maxillary agenesis (TAC 0.0.2.2/2.2.0.0) in 4.3% of ns-RS patients and left unilateral agenesis (TAC 0.2.0.0) in 1.7%. The maxillary lateral incisors were absent in 7 of 28 patterns and the mandibular lateral incisors in 5 of 28 patterns.Tables N = 36/55) of ns-RS patients with tooth agenesis, while unilateral agenesis was seen in 11 patterns or 34.5% (N = 19/55) of patients. A completely symmetrical pattern of tooth agenesis was noted in 6 patterns or 45.5% (N = 25/55) of patients with hypodontia. In maxillary agenesis, 60.7% (N = 17/28) of patients had a symmetrical pattern compared to 48.9% (N = 22/45) with mandibular tooth agenesis.Bilateral tooth agenesis was observed in 17 patterns or 65.6% (N = 10/57) of ns-CP patients with dental agenesis, which did not differ significantly from the situation in ns-RS as shown in Table P = 0.002). Figure As presented in Tables N = 30/57) of patients with tooth agenesis. Symmetrical patterns of tooth agenesis were seen in 4 patterns or 35.1% (N = 20/57) of patients with hypodontia. In patients with maxillary agenesis, 38.7% (N = 12/31) had a symmetrical pattern against 48.7% (N = 19/39) of patients with mandibular agenesis. Table In ns-CP, 12 patterns included bilateral agenesis translating into 52.6% (P = 0.539) and ns-CP (P = 0.947), there was no significant association between the extent of the palatal cleft and tooth agenesis.Table The objectives of this study were to determine the prevalence of tooth agenesis (excluding third molars) and its patterns in a Dutch population of patients with ns-RS and ns-CP and to test the hypothesis that tooth agenesis in ns-RS is mainly related to the developmental disturbances that also produced the palatal cleft seen in the majority of patients. The present study included 115 patients with ns-RS and observed tooth agenesis in 47.8% of patients. This number corresponds roughly to the prevalence rates for tooth agenesis in ns-RS previously found in Canada  (42.3%),In the comparison group of 191 patients with ns-CP, the prevalence of tooth agenesis was lower with 29.8%, which lies within the range of 21.0–36.8% described for ns-CP in other patient populations –19. AgaiWith regards to the patterns of hypodontia, this study showed that in line with the literature , 7, 20, IRF6, MSX1, PAX9, and TGFB3 [This study also examined the relationship between the extent of the palatal cleft and the degree of hypodontia. Developmental anomalies of the dentition are increasingly considered to be a subphenotype of the cleft population since both teeth and lip/palate are derived from the branchial arch precursors and influenced by related morphogenetic patterning signals . Indeed,nd TGFB3 , 9. Morend TGFB3 . HoweverSOX9 gene implicated in the non-syndromic form of the condition [In summary, this study showed a greater prevalence of tooth agenesis in ns-RS relative to ns-CP, a more pronounced mandibular agenesis in ns-RS, and the absence of a direct relationship between the extent of the palatal cleft and hypodontia in ns-RS. Consequently, additional (or even different) developmental disturbances are probably involved in tooth agenesis in ns-RS, with disturbances related to mandibular hypoplasia, the most likely candidates. The failure of mandibular outgrowth in ns-RS is hypothesized to result from intrauterine constraints on the mandible , defectsondition . Subphenondition , 32. TheThe strength of this study is the inclusion of a comparison group of patients with ns-CP and the use of the TAC system which precisely elucidated the differences in the prevalences and patterns of tooth agenesis between ns-RS and ns-CP. In addition, this study examined the relationship between the degree of hypodontia and the extent of the palatal cleft using formal statistical analysis. Despite these qualities, the study is subject to several limitations. First, it has a retrospective character without a strict case-control design. Second, the study did not assess tooth agenesis in the deciduous dentition.Tooth agenesis is more prevalent in ns-RS than that in ns-CP, demonstrates a much greater predilection for the mandible in ns-RS, and bears no relation to the palatal cleft. These findings suggest that additional developmental disturbances are likely involved in the etiology of tooth agenesis in ns-RS when compared to ns-CP. Future research could help identify the underlying genetic traits and aid in classifying patients in those with and without expected tooth agenesis in order to facilitate orthodontic management strategies.


Numerous genetic alterations predicting prognosis and clinical outcome are revealed recently in chronic lymphocytic leukemia (CLL). Among them the deregulated expression of micro RNAs that can induce tumor growth, or act as tumor suppressors seem to be of great importance. This study aimed to analyze the possible role of chosen micro RNAs as markers of prognosis in patients with CLL. We assessed the expression of miR-21, miR-34a, miR-181a, miR-199a/b and miR-221 in previously separated leukemic cells with the use of qRQ-PCR technique at the moment of diagnosis. The results were then analyzed in regards to presence of prognostic factors, clinical data and the end points like progression free survival (PFS), time to progression (TP) and overall survival time (OS).We detected significant correlations between expression of the analyzed micro RNAs and CLL prognostic markers particularly as far as miR-221 and miR-181a were concerned. The subsequent analysis revealed that high expression of miR-34a and miR-181a as well as low miR-21 expression indicated longer TTP, while miR-221 was predictor of OS.The obtained results prove the role of micro RNAs as CLL prognostic markers, particularly as factors predicting survival in a course of the disease. Chronic lymphocytic leukemia (CLL), one of the most frequently diagnosed leukemias, is characterized by the accumulation of leukemic CD19+/CD5+/CD23+ B cells in the blood, bone marrow, lymph nodes and spleen . NumerouLots of mechanisms involved in leukemic transformation of CLL are reported. The B cell receptor signaling plays an important pathogenic role because of BCR-dependent survival of leukemic lymphocytes , 9. Pro-The presented article aimed to analyze the possible role of chosen micro RNAs as a markers of prognosis in patients with CLL. The expression of miR-21, miR-34a, miR-181a, miR-199a/b and miR-221 in previously separated CD19+ leukemic cells was assessed with use of qRQ-PCR technique at the moment of diagnosis. The obtained results were then analyzed in regards to presence of prognostic factors, clinical data and the end points like progression free survival (PFS), time to progression (TP) and overall survival time (OS).Our analysis revealed an expression of all analyzed micro RNAs in leukemic cells except of miR-199a/b, so this micro RNA was not further assessed. Micro RNAs expression presented as mean ± standard deviation are shown in Table We detected statistically significant negative correlation between expression of miR-221 and both leukocytosis and lymphocytosis. Other micro RNAs were not in correlation with parameters of complete blood count. The expression of both miR -181a and miR-221 were in adverse significant correlation with serum level of β2-mikroglobulin. These micro RNAs were also in negative correlation with expression of CD38 on leukemic cells, however such a relationship was not found as far as expression of ZAP-70 was concerned. Additionally, expression of miR-221 was detected to be different in regard to cytogenetic risk. In a group of standard-cytogenetic risk the expression was higher in comparison to high-risk group. The results are shown in Figure The group of studied patients was assessed in regards to first line chemotherapy regimens as well as chemotherapy outcome. Only the subjects (20 persons) who required therapy because of the disease progression were enrolled into this part of study. The following schemes of chemotherapy were used: chlorambucil + prednisone (9 persons), fludarabine + cyclophosphamide (7 persons), fludarabine + cyclophosphamide + rituximab (2 persons), bendamustine (1 person), cyclofosfamide + vinkristin + doksorubicin + prednisone (1 person). The patients were classified as responders  and non-responders (those who have stable disease or progression). Statistical analysis indicated that expression of analyzed micro RNAs was not a predictor of chemotherapy outcome, however the variability of regimens used and a low number of patients in each group might influence the results.n = 14; 1.93 ± 3.97 months ) and the second one with time to progression longer than 1 year after beginning of leukemia . Analysis of micro RNAs expression in the above groups revealed the statistically significant difference in expression of miR-34a. Similarly, Kaplan–Meier analysis of progression probability showed statistically significant difference between a group of patients with high versus low expression of miR-34a. These results are shown in Figure F = 7.79; p < 0,001; ∆R2 = 0.59). High expression of miR-34a (β = 0.67) and miR-181a (β = 0.49) as well as low miR-21 expression (β = –0.46) significantly influence TTP. The data are presented in Figure The study group was analyzed with reference to time to leukemia progression. In the moment of analysis 20 out of 40 enrolled patients were diagnosed because of leukemia progression. The others were in stable phase of the disease. The patients with progression detected were divided into 2 groups: the first one with moment of progression which occurred within 1 year after diagnosis  and the second one who progressed in the period longer than one year . Analysis of microRNA in the above groups revealed statistically significant difference only in case of miRNA-221 expression . Analysis based on linear regression in regards to all studied micro RNAs showed no statistical significance . Assessment with Kaplan–Meier test showed no statistically significant difference in predicting PFS between groups with high and low micro RNA expression, respectively.The analysis of PFS was performed in the group of subjects who have completed first-line therapy (20 persons). They were divided into two groups: the first one of patients who progressed after the first line of treatment within one year (n = 19) died. All subjects were divided into two groups: the first one - patients who survived less than 5 years  and the second one -those who survived more than 5 years . Analysis of micro RNAs in the above groups revealed statistically significant difference in miRNA-221 expression. The linear regression assessment did not revealed statistical significance . Analysis with Kaplan–Meier test showed that the probability of survival was higher in the group of patients with elevated level of miR-221 expression in comparison to the group with lower miR-221 level, however the difference was not statistically significant . Up to the moment of analysis 45.2% of patients  miR-221 expression was significantly lower than in those with standard cytogenetic risk. Additionally, we detected negative correlation between miR-221 expression and β-2-mikroglobulin level. It can be concluded that a low expression of this micro RNA results in more progressive course of leukemia and is correlated with the presence of high-risk-cytogenetic profile. In case of miR-181a the correlation with CD38 expression was detected. In patients with low miR-181 level the expression of CD38 was significantly higher. Similarly, in patients with higher β-2-mikroglobulin concentration the expression of miR-181a was significantly lower. There were no detectable difference between expression of other micro RNAs and prognostic factors. Expression of miR199a/b was not detected at any of studied patients indicating that this miRNA cannot be consider as prognostic factor in CLL.et al. [et al. [et al. [et al. [et al. [et al. [The literature data on micro RNA expression in course of CLL are incomplete and sometimes confused. Rodrigez et al.  showed t [et al.  indicate [et al. –25. Trop [et al.  assessed [et al.  showed l [et al.  analyzed [et al. . The rol [et al. . Similar [et al. . Rossi e [et al.  indicate [et al. .et al. [et al. [Our analysis of micro RNAs expression in regards to end points in CLL patients revealed that high expression of miR-34a and miR-21 as well as low miR-181a expression significantly influenced TTP, while miR-221 and miR-181a indicated OS. Some literature data showed correlations of microRNA expression and the end points in hematologic disorders. Marcuccii et al.  indicateet al. . In DLBCet al. . Zhu et  [et al. –37. Ther [et al. , 39, how [et al. . In rega [et al. , 42. The [et al. , 21.et al. [et al. [The group of studied patients was assessed in regards to chemotherapy outcome, however no significant correlations were detected. Scientific literature data indicated that expression of specific micro RNAs may help to predict the chemotherapy outcome in CLL patients. Moussay et al.  showed t [et al.  proved t [et al. , 46. In To summarize, based on our results and the presented literature data we can conclude that micro RNAs may be the prognostic factors in the course of the disease and the predictors for end points in CLL patients. Expression of miR-21, miR-34a and miR-181a may be useful in predicting TTP, while expression of miR-221 may indicate OS. Thus further studies are required in this field of CLL biology to prove the importance of micro RNAs expression in clinical practice.Forty newly diagnosed CLL patients, previously not treated, were enrolled into the study. The diagnosis of CLL was based on clinical examination and morphological and immunological criteria. The clinical characteristics are shown in Table Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation . After washing with phosphate-buffered saline, the number and viability of cells were assessed with trypan blue staining. Viability below 95% was a disqualifying criterion for further study. PBMC were subsequently subjected to the procedure of leukemic cells separation. The magnetic activated cell sorting method was used according to manufacturer’s instruction . PBMC were firstly incubated with magnetic beads coated with monoclonal antibody (MoAb) anti-CD19 in MACS buffer and then subjected to washing procedures to obtain the cell pellet for application to the MACS column. In this way the CD19+ leukemic cells were obtained, their viability was assessed and they were used for further procedures.Total RNA was isolated from leukemic cells according to the modified method of Chomczynski and Sacchi . The mir® Small RNA Assays Kit . Initially, total cellular RNA was reverse-transcribed with specific primers using TaqMan®MicroRNA Reverse Transcription Kit according to the manufacturer’s protocol on Applied Biosystems 7500 Fast Real Time PCR Systems. The obtained complementary DNA (cDNA) was used for further procedures. The quantitative analysis of microRNA expression was done with quantitative reverse transcriptase real-time PCR, (qRT-PCR) method. Following probes were used: hsa-miR-21-5p, hsa-miR-34a-5p, hsa-miR-181a-5p, hsa-miR-199a-5p, hsa-miR-221-3p and hsa-miR-16 as endogenous control. A positive reaction was detected by accumulation of a fluorescent signal. The cycle threshold (Ct) was defined as the number of cycles required for the fluorescent signal to cross the threshold and exceeds background level. Ct levels were inversely proportional to the amount of microRNA in the sample. The expression of each micro RNA was normalized with the endogenous control. The comparative ΔΔCq method was then applied for data analysis, and fold changes were next calculated using 2–ΔΔCq [Assessment of microRNA expression was performed using the TaqMang 2–ΔΔCq . All PCR® 488 Mouse IgG2a Labeling Kit  was added to the sample tubes. The samples were incubated for 30 min, washed, and examined by flow cytometry method. When ZAP-70 expression was detected in ≥ 20% of leukemic cells, the subject was considered positive for ZAP-70. To assess CD38 expression, PBMC were stained with anti-CD38 FITC, anti-CD19 PE, anti-CD5 CyChrome MoAbs, or IgG1 isotypic control (BD Pharmingen) for flow cytometry analysis. Patients were considered CD38 positive when expression was found in at least 20% of CLL cells.Sample of PBMC cells were stained with the MoAbs anti-CD19 PE-Cy7, CD5 APC or CD3 PE (BD Pharmingen). After membrane staining, the cells were fixed by 1% paraformaldehyde solution. Next, anti-ZAP-70 antibody  that was labeled by the ZenonTM Alexa Fluorin situ hybridization (FISH) method was used to determine cytogenetic abnormalities in leukemic cells. The locus-specific probes 17p13.1 (LSI TP53), 11q22.3 (LSI ATM), 13q14.3 (D13S319), 13q34, and the chromosome 12 centromere  were used. Procedures were performed according to the manufacturer’s protocol. Probes were denatured at 73°C for 5 min and then applied to the designated areas of the slides. Following an overnight hybridization the slides were stained with DAPI. The analysis was performed using a BX51 fluorescence microscope , and CytoVision image analysis system. At least 200 nuclei were assessed for each probe, and the border value for positive result was 20%.Fluorescence In the group of subjects enrolled into the study the following clinical data were analyzed: time from diagnosis to starting of the therapy (time to progression-TTP), chemotherapy regimens ordered and chemotherapy outcome , probability of progression free survival (PFS) and probability of overall survival (OS). The criteria of therapy response proposed by WG- IWCLL in 2008  based onp < 0.05 was considered to be statistically significant.Statistical analysis was performed with STATISTICA 12.0 software for Windows. The results were shown as median or mean values with standard deviation. The Mann–Whitney and Wilcoxon tests were used for groups comparison. The Kaplan–Meier method was employed to calculate the survival analysis. Multivariate analysis of independent clinical factors for survival was tested by linear regression. Value of


Assessment of color disc photograph (C-DP) is affected by image quality, which decreases the ability to detect glaucoma. High-dynamic-range (HDR) imaging provides a greater range of luminosity. Therefore, the objective of this study was to evaluate the capability of ophthalmology residents to detect glaucoma using HDR-concept disc photography (HDR-DP) compared to C-DP.  Cross-sectional study.  Twenty subjects were classified by 3 glaucoma specialists as either glaucoma, glaucoma suspect, or control. All C-DPs were converted to HDR-DPs and randomly presented and assessed by 10 first-year ophthalmology residents. Sensitivity and specificity of glaucoma detection were compared. μm, 100.2 ± 9.6 μm, and 105.8 ± 17.2 μm for glaucoma, glaucoma suspect, and controls, respectively. The diagnostic sensitivity of HDR-DP was higher than that of C-DP . Regarding diagnostic specificity, HDR-DP and C-DP yielded 46% and 75% .  The mean ± SD of averaged retinal nerve fiber layer (RNFL) thickness was 74.0 ± 6.1  HDR-DP statistically increased diagnostic sensitivity but not specificity. HDR-DP may be a screening tool for nonexpert ophthalmologists. Glaucoma is a chronic progressive optic neuropathy that is characterized by loss of retinal nerve tissue and field of vision. Due to the asymptomatic nature of the disease, most patients are not aware that they have glaucoma until the late stage of the disease. Currently, more than 8.4 million people worldwide are bilaterally blind from glaucoma . It has This cross-sectional study was conducted at the Department of Ophthalmology, King Chulalongkorn Memorial Hospital  and was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University. This study was conducted in accordance with the Declaration of Helsinki and all of its subsequent amendments. There were 3 groups of participants in this study, including glaucoma patients, glaucoma suspect patients, and healthy volunteers. Color optic disc photographs were taken from the eyes of participants in all 3 subgroups. Evaluators consisted of 10 first-year ophthalmology residents. After receiving permission to access and retrieve images, color disc photographs of glaucoma and glaucoma suspect patients were recruited from our hospital's imaging database. Written informed consent was obtained from healthy volunteers prior to their participation in the study. All subjects were more than 18 years old and had a spherical refractive error between −6 diopters and +6 diopters and astigmatism of less than 3 diopters. Exclusion criteria were history of intraocular trauma, retinal disease, neurological disease, and uncooperative subject. All glaucoma and glaucoma suspect subjects had undergone C-DP , optical coherence tomography (OCT) , and standard automated perimetry (SAP)  within 6 months prior to the start of the study. All C-DPs from the glaucoma and glaucoma suspect groups were performed in dilated condition by a single experienced photographer at dimensions and settings of 1600 × 1216 pixels and RGB color space in JPEG format. Only qualified images were recruited. Glaucoma patient was defined as vertical cup-to-disc ratio greater than 97.5th percentile of normal population with presence of RNFL defect in OCT or disc photograph that correlated with visual field defect in SAP. Presence of vertical cup-to-disc ratio between 97.5th and 99.5th percentiles of normal population without RNFL defect or functional visual field loss and IOP of less than 21 mmHg were classified as glaucoma suspect subjects. Healthy volunteers who participated in this study had visual field tests and images taken following the same process by the same single photographer. Healthy participants had a vertical cup-to-disc ratio of less than 97.5th percentile with a normal reliable SAP result. Nonqualifying images, OCT, and visual fields included the following: (1) images with poor visualization of retinal blood vessels; (2) OCT scans with incorrect ONH detection, off-centered ONH, motion artifacts, wrong segmentation, or signal strength less than 6; and (3) unreliable SAP with fixation loss, false-positive or false-negative responses greater than 20%. Images from 10 glaucoma patients, 5 glaucoma suspect patients, and 5 healthy volunteers were taken, recorded, and included in the study. A total of 20 C-DPs from all subjects were recruited from April to October 2014. Three glaucoma specialists  evaluated C-DP, OCT, and SAP to confirm the diagnosis and to identify the number and affected areas of peripapillary RNFL defect in six quadrants  . The detAll recruited C-DPs were then processed into HDR photos by adjusting the light exposure as overexposed, normally exposed, or underexposed .Image editing was performed using preview program for Mac OS X version 8.0. Three different exposure images were then combined using the following settings: strength −4.4, brightness 0.4, local contrast 8.4, white Clip 1.0, black Clip 0, midtone 0, and color saturation −1.5 .After that, the ten first-year ophthalmology residents who volunteered to join the study provided written informed consent to participate as evaluators. All evaluators were trained in how to diagnose glaucoma and identify RNFL defect. Evaluators were blinded to patient clinical information and diagnosis. The ophthalmology resident evaluators independently assessed a set of 40 randomly sequenced disc photographs consisting of 20 C-DPs and 20 HDR-DPs. Images were projected onto a projector screen at a resolution of 1280 × 800 pixels with 32-bit color in a dark room, and all of the evaluators assessed the image within a period of 30 seconds. The residents were asked to answer 2 questions within 30 seconds, as follows: determine whether the presenting image is glaucoma or not and identify the number and location of RNFL defect.t-test was used to test statistical differences of sensitivity and specificity between C-DP and HDR-DP, as evaluated by the residents when using the glaucoma specialists' references. The primary outcomes were mean difference ± SD of sensitivity and specificity of the test. p values less than 0.05 were considered statistically significant.All statistical analyses were performed using SPSS version 17.0 . Paired Demographic and clinical data of study subjects are shown in All subjects had best-corrected visual acuity better than 20/30. Distribution of sensitivity and specificity of glaucoma diagnosis using C-DP and HDR-DP by the 10 ophthalmology residents is shown in The average sensitivity of glaucoma detection in HDR-DP was better than that in C-DP . The mean difference of sensitivity was 19.0 ± 19.7 (95% CI: 4.91 to 33.1). The average specificity of glaucoma detection in HDR-DP and C-DP was 46% ± 28.8% and 75% ± 17.2%, respectively. The mean difference in specificity was 29.0 ± 21.8 (95% CI: 13.4 to 44.6) .The average sensitivity of RNFL defect detection between C-DP and HDR-DP in each quadrant of the glaucoma group was analyzed, but no statistical significant difference was observed . InterobIn this study, we applied the HDR technique and evaluated diagnostic accuracy compared with original C-DP in nonexperienced trainees. We found that HDR-DP had higher sensitivity for detecting glaucoma than C-DP, but specificity for detection of glaucoma using HDR-DP was lower than that using C-DP. RNFL defect detection was similar between the two techniques.Color disc photograph is the backbone diagnostic method for glaucoma diagnosis and is widely used in clinical practice. To improve the visualization of optic disc characteristics, stereoscopic disc photograph for three-dimensional evaluation is normally recommended. Many studies have compared the diagnostic ability of using stereoscopic versus monoscopic disc photographs to detect multiple optic disc characteristics, and they have reported varying and sometimes controversial results , 14. A rTo highlight RNFL visualization, red-free disc photograph and blue reflectance RNFL with confocal scanning laser ophthalmoscopy were assessed and compared with C-DP . They foHowever, HDR-DP had lower specificity than C-DP, as well as average sensitivity for RNFL defect in each quadrant in the glaucoma group. This finding was possibly due to the higher false-positive rate among normal disc photographs and glaucoma suspect photographs. Both slit defect mimicking true wedge-shaped RNFL defect and unrecognized defect in diffuse RNFL loss images could be the explanation for high false-positive results. Differences between the HDR-DP and C-DP techniques for RNFL defect detection in each quadrant were not observed. This may be explained by the fact that HDR potentially improved visualization of not only the RNFL but also the disc characteristics, which may have led to the bias of RNFL defect at the corresponding disc area.Interobserver agreement in our study was 0.33 (95% CI: 0.21 to 0.45) and 0.43 (95% CI: 0.31 to 0.56) for HDR-DP and C-DP, respectively. This was similar to Callewaert et al. that reported interobserver variability on fundus images of 0.29 for trainees and 0.37 for residents . Even afThere were some limitations in our study. First, low specificity was found due to overestimation of RNFL defect in the HDR-DP group. The RNFL pattern in HDR-DP of normal and glaucoma suspect patients mimicked slit RNFL defect and may have induced false-positive results. Improvement in HDR program parameters may improve overall specificity and increase sensitivity of RNFL defect, when compared to C-DP. Secondly, we performed the study using first-year residents that had only 3 months of clinical ophthalmology training. Although we had briefly trained them on how to evaluate optic disc photograph in glaucoma, their lack of experience remained the predominant drawback. In addition, the residents may have had added difficulty with HDR interpretation, because this was the first time HDR concept was applied to disc photograph. These factors resulted in the high interobserver variations found in our study. Further study is needed using evaluators with different levels of experience. Moreover, we did not explore other specific characteristics of the optic disc that affect or may affect glaucoma detection. These optic disc characteristics should also be further investigated.In summary, HDR-DP provided better sensitivity for glaucoma detection than C-DP, but specificity was not improved. HDR-DP might be an effective alternative glaucoma screening tool for general practitioners, nonexpert ophthalmologists, and trainees.


Vibrio alginolyticus is herein described. The DNA nanostructure-modified magnetic beads are used for amplification of the potential response and elimination of the interfering effect from a complex sample matrix. The solid-contact polycation-sensitive membrane electrode using protamine as an indicator is employed to chronopotentiometrically detect the change in the charge or DNA concentration on the magnetic beads, which is induced by the interaction between Vibrio alginolyticus and the aptamer on the DNA nanostructures. The present potentiometric aptasensing method shows a linear range of 10–100 CFU mL−1 with a detection limit of 10 CFU mL−1, and a good specificity for the detection of Vibrio alginolyticus. This proposed strategy can be used for the detection of other microorganisms by changing the aptamers in the DNA nanostructures.A potentiometric aptasensing assay that couples the DNA nanostructure-modified magnetic beads with a solid-contact polycation-sensitive membrane electrode for the detection of  Vibrio alginolyticus  can not only lead to septicemias and corneal opaqueness in fish and shell diseases and white spots in shrimps [V. alginolyticus is of great importance for aquaculture, food industry, and clinical diagnosis.As an opportunistic marine pathogen,  shrimps ,2,3, but shrimps ,5. The rThe agar plate culture method and enzyme-linked immunosorbent assays are commonly used for the detection of bacteria. However, they suffer from such problems as time-consuming procedures, false-positive signals, low sensitivity, and poor specificity . The polListeria monocytogenes in a homogeneous solution [Aptamers are single-stranded DNA or RNA oligonucleotides designed through the systematic evolution of ligands by exponential enrichment. Since aptamers have high specificities and binding affinities to their targets, they have been widely used to develop a variety of electrochemical biosensors for the detection of small molecules, metal ions, and proteins ,13,14,15solution , based oV. alginolyticus based on DNA nanostructure-modified magnetic beads for signal amplification and magnetic separation, and on a solid-contact polycation-sensitive membrane electrode for reversible potential detection via chronopotentiometry. In the presence of V. alginolyticus, the DNA nanostructure-modified magnetic beads can be disassembled, which is induced by the specific binding interactions between the target bacterial cells and the aptamer molecules in the DNA nanostrutures. After magnetic separation, the change in the charge or DNA concentration on the magnetic beads can be chronopotentiometrially detected by the solid-contact polycation-sensitive electrode using protamine as an indicator based on the electrostatic interaction between DNA and protamine.Herein, we present a novel potentiometric aptasensing strategy for the detection of o-NPOE), tetradodecylammonium chloride (TDDACl), dinonylnaphthalene sulfonic acid , tris (hydroxymethyl)-aminomethane (Tris), protamine sulfate salt, and ionic liquid  were purchased from Sigma-Aldrich. Carboxylic-multiwall carbon nanotubes (MWCNTs) were obtained from Nanjing XFNANO Materials Technology Co. Ltd. . The lipophilic salt DNNS-TDDA was synthesized as described before [V. alginolyticus 1.1587 and Aeromonas hydrophila 1.172 were purchased from Yudingxinjie Technology Co. Ltd. . Bacterial strains for Escherichia coli (E. coli) ATCC 27853 and Staphylococcus aureus 08032813 were kindly provided from the Biotechnology Lab of Binzhou Medical University. The number of colony-forming units per mL (CFU mL−1) for each bacteria culture was determined by the surface plate counting method. The aptamer used in this work for the detection of V. alginolyticus was selected by Zheng et al. [2 (pH = 7.4).High-molecular-weight poly(vinyl chloride) (PVC), 2-nitrophenyl octyl ether (d before . The mag−1) via shaking for 0.5 h. After magnetic separation and washing with buffer, the capture DNA modified magnetic beads were incubated with 150 μL of aptamer DNA fragments (3 μM) via shaking for 1 h, and the capture/aptamer DNA modified magnetic beads were thus obtained. The H1/H2 DNA complexes were prepared by incubating 150 μL of H1 DNA fragments (3 μM) with 150 μL of H2 DNA fragments (3 μM) via shaking for 1 h. The H1/H2 DNA complexes (150 μL) were then incubated with 150 μL of capture/aptamer DNA modified magnetic beads with 1 h shaking. After magnetic separation and washing with buffer, the DNA nanostructure-modified magnetic beads were obtained and re-dispersed in 150 μL of buffer solution. For the detection of bacteria, 40 μL of the prepared DNA nanostructure-modified magnetic bead solution (10 mg·mL−1) were used.All the oligonucleotides were kept at 95 °C for 2 min and then cooled at room temperature for 1 h before use ,26,27. TPrior to modification, the glassy carbon electrode  was polished with emery paper and alumina slurries followed by rinsing thoroughly with ultrapure water. The electrode was successively ultrasonicated in water and ethanol, and then allowed to dry at room temperature.−1 suspension solution. Twenty microliters of the MWCNT-IL suspension solution was coated onto the surface of the GCE and allowed to dry under an infrared lamp.A mixture of 5 mg of MWCNTs and 1 μL of IL was ground in an agate mortar for 10 min , and theo-NPOE, and 30.0 wt % PVC. One hundred milligrams of the membrane components were dissolved in 800 μL of tetrahydrofuran (THF). Eighty microliters of the membrane solution were coated onto the MWCNT-IL modified GCE. After the evaporation of THF, the prepared solid-contact polycation-sensitive electrodes were conditioned in 10 mM NaCl for at least 12 h before use, and kept in the conditioning solution when not in use.The polycation-sensitive membrane contained 10.0 wt % DNNS-TDDA, 60.0 wt % V. alginolyticus at different concentrations at room temperature for 30 min. After magnetic separation, the resulting magnetic beads were then incubated with 7.5 μL of 1 mg·mL−1 protamine. The unreacted protamine after magnetic separation was added into 1.5 mL of 0.01 M NaCl and detected by the solid-contact polycation-sensitive electrode via chronopotentiometry.Forty microliters of DNA nanostructure-modified magnetic beads were mixed with 50 μL of V. alginolyticus was used for quantification.Chronopotentiometry was performed by using a CHI-760C electrochemical workstation  with a conventional three-electrode system comprising the proposed solid-contact polycation-sensitive membrane electrode as a working electrode, a platinum wire as counter electrode, and an Ag/AgCl electrode (saturated KCl) as a reference electrode. During the chronopotentiometric experiments, an external cathodic current of 5 μA with a duration of 1 s was used to extract protamine into the polycation-sensitive membrane. The solid-contact polycation-sensitive membrane electrode was refreshed under a controlled voltage at the open-circuit potential in the absence of protamine with a recovery time of 180 s for multiple consecutive measurements. The difference between the potentials measured at 0.5 s after applying the current in the absence and presence of V. alginolyticus based on DNA nanostructure-modified magnetic beads. The capture DNA was immobilized on the surface of the magnetic beads via the strong streptavidin-biotin interaction. The aptamer and H1/H2 DNA hybridize successively with the capture DNA through the complementary base-pairing reactions to form the DNA nanostructure-modified magnetic beads. In the presence of the target, the aptamer on the DNA nanostructure-modified magnetic beads specifically binds to the target, which leads to the disassembly of the DNA nanostructures and subsequently influences the charge or DNA concentration on the magnetic beads [ic beads . This apV. alginolyticus, the effect of protamine concentration was investigated. −1, the potential change is up to 30 mV. Considering the subsequent electrostatic interaction between the DNA nanostructures and protamine and the sensitivity of the polycation-sensitive membrane electrode, 5 μg·mL−1 protamine was selected for further experiments.Since protamine was used as an indicator to detect the change in the DNA concentration on the DNA nanostructure-modified magnetic beads induced by The influence of the amount of the DNA nanostructure-modified magnetic beads on the potential response to protamine of the solid-contact polycation-sensitive membrane electrode was investigated. As shown in V. alginolyticus were tested by using the solid-contact polycation-sensitive membrane electrode via chronopotentiometry. As shown in V. alginolyticus increases with increasing the concentration of V. alginolyticus, which indicates that the disassembly of the DNA nanostructures results in the decrease in the charge or DNA concentration on the surface of the modified magnetic beads. −1, and the detection limit is 10 CFU mL−1.Under the optimal experimental conditions, the potentiometric responses to V. alginolyticus, four bacterial strains including V. alginolyticus, E. coli, Staphylococcus aureus, and Aeromonas hydrophila were tested. As shown in V. alginolyticus has a good specificity. Additionally, the influence of the random DNA on the potential response was also investigated (In order to test the specificity of the potentiometric aptasensing method for the detection of stigated B. The reV. alginolyticus. The proposed method shows a linear concentration range of 10–100 CFU mL−1 with a detection limit of 10 CFU mL−1, and a good specificity for the detection of V. alginolyticus. The proposed strategy can be used for the detection of other microorganisms by changing the aptamers in the DNA nanostructures.This work demonstrates a potentiometric aptasensing assay using signal amplification and magnetic separation strategies. The method is based on the DNA nanostructure-modified magnetic beads and the solid-contact polycation-sensitive membrane electrode for the reversible chronopotentiometric detection of


Dundee Ready Educational Environment Measure (DREEM) is a 50-item tool to assess the educational environment of medical institutions as perceived by the students. This cross-sectional study developed and validated an abridged version of the DREEM-50 with an aim to have a less resource-intensive , yet valid and reliable, version of DREEM-50 while also avoiding respondent fatigue.n = 418) from a private teaching hospital in Madurai, India, were divided into two groups. Group I (n = 277) participated in the development of the abridged version. This was performed by domain-wise selection of items that had the highest item-total correlation. Group II (n = 141) participated in the testing of the abridged version for construct validity, internal consistency and test-retest reliability. Confirmatory factor analysis was performed to assess the construct validity of DREEM-12.A methodology similar to that used in the development of WHO-BREF was adopted to develop the abridged version of DREEM. Medical students (p < 0.001). Confirmatory factor analysis of DREEM-12 construct was statistically significant (LR test of model vs. saturated p = 0.0006). The internal consistency of DREEM-12 was 0.83. The test-retest reliability of DREEM-12 was 0.595, p < 0.001.The abridged version had 12 items (DREEM-12) spread over all five domains in DREEM-50. DREEM-12 explained 77.4% of the variance in DREEM-50 scores. Correlation between total scores of DREEM-50 and DREEM-12 was 0.88 (DREEM-12 is a valid and reliable tool for use in educational research. Future research using DREEM-12 will establish its validity and reliability across different settings. Education environment refers to the diverse physical locations in which the students learn. The educational environment of an institution has direct and indirect influence(s) upon the knowledge gained by the students including the motivation to learn, personal safety and their well-being . Dundee n = 12), students’ perception of teachers , students’ academic self-perception , students’ perception of atmosphere , and students’ social self-perception . It is a generic and culturally non-specific tool which has been translated and validated in at least eight languages and is used worldwide [DREEM has 50 items grouped under five domains namely students’ perception of learning , yet valid and reliable, version of DREEM-50 while also avoiding respondent fatigue.A cross-sectional study was conducted for development of an abridged version of DREEM at a private, tertiary care teaching institute in Madurai, a city in Tamil Nadu state of India. It is one of the two major and the only private institution teaching medicine and allied health sciences in the city. Every year, 150 students are selected through a competitive examination for the undergraduate course in medicine—Bachelor of Medicine and Bachelor of Surgery .n = 300) and group II .A list of the 450 undergraduate medical students currently enrolled in the institute  as on August 2016 was obtained, and all students were invited to participate in the study. These students were divided into two groups group I . Items that had to be reverse coded were done so before entry. The data was analysed using STATA . Number and proportion were calculated for sociodemographic characteristics like gender and semester. Mean and standard deviation were calculated for age and for score of each item, domain and total scale of DREEM-full and abridged version.For the development of the abridged version, the authors followed a procedure similar to that adopted in the development of WHOQOL-BREF, an abridged version of WHOQOL-100, which measures quality of life (QOL) . Item-tor).DREEM-50 and DREEM-12 scores were calculated on group II dataset. Internal consistency was measured using Cronbach’s alpha. The percentage of variance in DREEM-50 that is explained by the abridged version was calculated using R square. DREEM-12 alone was administered on group II to get the second set of scores of DREEM-12 to check for test-retest reliability , i.e. SPoT (two), SPoT (three), SASP (three), SPoE (two) and SSP (two).Ethics approval was obtained from institute ethics committee of Velammal medical college hospital and research institute, Madurai, Tamil Nadu. Written informed consent was taken from the students and the consent process was approved by the ethics committee.A total of 277 students from group I participated in the development of the abridged version and 141 students from group II in the validation and reliability of the abridged version. The age and gender distribution of the study population is given in Table p < 0.001). Domain–domain correlation between DREEM-50 and DREEM-12 were 0.75 (SPoL), 0.7 (SPoT), 0.79 (SASP), 0.66 (SPoA) and 0.62 (SSP). Regression analysis revealed that DREEM-12 explained 77.4% of the variance in DREEM-50. The internal consistency of DREEM-12 was 0.83 as compared to 0.86 of DREEM-50.The item-total correlation and domain-total correlation of the items selected for inclusion in DREEM-12 are given in Table p < 0.001.The item wise and overall scoring system of DREEM-12 was devised in line with the DREEM-50 Table . When caχ2(44) = 80.88, Prob> χ2 = 0.0006) . This study failed to propose an abridged version (33 items) mentioning two reasons, namely, the weakest items may not be same for different settings and fear of affecting the factor structure .Reports on test-retest reliability of DREEM are variable. A study from Brazil reported moderate test-retest reliability of 0.43 . ContrarDREEM-12, developed as an abridged version of DREEM-50, was found to explain 77.5% of the variance of the scores of the original version and also retained its factor structure, validity and reliability. Future research using DREEM-12 will establish its validity and reliability across different settings.


In positron emission tomography (PET) studies, thoracic movement under free-breathing conditions is a cause of image degradation. Respiratory gating (RG) is commonly used to solve this problem. Two different methods, i.e., phase-gating (PG) and amplitude-gating (AG) PET, are available for respiratory gating. It is important to know the strengths and weaknesses of both methods when selecting an RG method for a given patient. We conducted this study to clarify whether AG or PG is preferable for measuring fluorodeoxyglucose (FDG) accumulation in lung adenocarcinoma and to investigate patient conditions which are most suitable for AG and PG methods.max) of PG, AG, and the control data of these two methods were measured, and the increase ratio (IR), calculated as IR(%)= (Post – Pre)/Pre × 100, was calculated. The diameter and position of lung lesions were also analyzed. We defined an ‘effective lesion’ of PG (or AG) as a lesion which showed a higher IR compared to AG (or PG). 8 (25.8%)A total of 31 patients  with 44 lung lesions, diagnosed as lung adenocarcinoma between April 2012 and March 2013, were investigated. Whole-body FDG-PET/CT scan was performed with both PG and AG methods in all patients. The maximum standardized uptake value , the average IR of PG was significantly higher than that of AG (P<0.01). The number of PG- and AG-effective lesions was 32 (72.7%) and 12 (28.3%), respectively. There was no significant difference in the average diameter or position of the lesions between the PG- and AG-effective lesions. There were 23 (74.2%) PG-effective and 8 (25.8%) AG-effective patients. No significant difference was observed in sex or age between PG- and AG-effective patients.The average SUVThe PG method was more effective for measuring FDG accumulation of lung lesions under free-breathing conditions in comparison with the AG method. Thoracic movement under free-breathing conditions is a cause of image degradation in positron emission tomography (PET) studies . RespiraRespiratory-gating (RG) methods have been recently introduced to solve the discussed problem. Phase-gating (PG)  and amplThe PG method is often used with respiratory tracking systems to monitor the respiratory motion during PET scans. The PET data are sorted into discrete bins, corresponding to certain time intervals in the respiratory cycle. The PET images, reconstructed from each of the bins, correspond to a phase of the cycle. In the AG method, the PET data are sorted into bins according to the respiratory amplitude range. This range is determined by analyzing the respiratory amplitude histogram.Kawano et al. suggested that breath-holding could effectively result in an improvement in image degradation  however,Several studies have demonstrated the advantages of RG in free-breathing acquisition using the PG -8 or AG In this study, we applied both methods for the same patients in a single FDG-PET/CT study. Our two main objectives were to establish which method is more effective in the measurement of FDG accumulation in lung adenocarcinoma and to clarify which patient conditions are suitable for PG or AG.A total of 31 patients  with 44 histopathologically confirmed lung adenocarcinomas, examined between April 2012 and March 2013, were evaluated in this study. All patients underwent WB FDG-PET/CT scan with PG and AG methods.The lung lesions were classified as either an “upper-lung lesion,” i.e., a lesion located at a level higher than the carina, or a “lower-lung lesion,” i.e., a lesion located at a level lower than the carina. There were 16 upper-lung lesions and 28 lower-lung lesions. The diameter of each lesion was manually measured in the axial diagnostic CT image, using the Response Evaluation Criteria in Solid Tumors (RECIST) methods . The chaPET data were acquired for all patients, using a Biograph mCT system . This PET scanner comprises of three rings with a total of 144 lutetium orthosilicate detectors, covering an axial field of view (FOV) of 16.2 cm and a transaxial FOV of 70 cm in diameter; each block is 4×4×20 mm.The coincidence time window was 4.1 ns, and the time of flight (TOF) time resolution was 555 ps. The spatial resolution values at 1 and 10 cm were 4.4 and 4.9 mm, respectively. The emission data were acquired in the 3D mode and reconstructed with a 256×256 matrix (3.18×3.18×5.00 mm). The 32-slice CT scan parameters were as follows: 120 kV, 100 mAs (Eff. mAs), a 512×512 matrix, slice thickness of 3 mm, and a transaxial FOV of 500 mm.18F-FDG administration. The injected activity of FDG was four times the patient’s body weight and 251.1±48.2 MBq on average.PET images were reconstructed using 3D ordered-subset maximization (3D-OSEM) with a point spread function (PSF) and TOF algorithm. The reconstruction parameters were as follows: two iterations, 21 subsets, and a post-filter of 5 mm full-width at half-maximum (FWHM) with CT attenuation correction. All patients fasted for at least 4 h before 18F-FDG. First, the WB scan, including the AG method, was performed accompanied with WB–CT scan for attenuation correction. Second, a PG study was performed in the same bed position as the AG study after 15 min of WB scan, followed by lung CT for attenuation correction. The acquisition time of each bed position including lung lesions was 8 min for the AG-PET study and 10 min for the PG-PET study.The schedule of the PET scan is summarized in The sequential 2-min acquisition data in the middle time point of PG or AG acquisition time were used as the control data. WB and chest CT scans for attenuation correction were performed between WB and PG scans. The average effective dose from FDG-PET was 4.7±0.9 mSv , 14, andThe chest wall pressure of the patients was sensed by a respiratory monitoring system  during PET data acquisition. The signals of chest wall pressure were converted to a respiratory wave in this system. The PG method is summarized in max) of the lesions was selected for the PG data. The number of selected bins is summarized in Each respiration cycle was divided into five gates as 20% of the cycle phase, and gates of the same number were combined for all respiratory cycles as a phase data (bin). The bin with the highest maximum standardized uptake value  was calculated as follows:The SUVIR (%)= (Post-Pre)/Pre×100max of the lesion in PG or AG, and ‘pre’ indicates the SUVmax of the lesion in the control data of the PG or AG method.where ‘post’ denotes the SUVWe defined a PG (or AG) ‘effective lesion’ as a lesion which showed a higher IR compared to AG (or PG). In addition, we defined ‘PG (or AG)-effective patients’ as those who had a PG- or AG-effective lesion, respectively. For patients with multiple lesions, the lesion which had the highest IR of all lesions was used for the calculation of IR in PG and AG to define PG- and AG-effective patients.Statistical analysis was performed using the statistical software JMP 11 . Wilcoxon signed-rank test was used to compare parameters between the control and gated data. Mann-Whitney U test was used to compare lesion diameter and age between PG- and AG-effective lesions. Fisher’s exact test was used to compare lesion position in the lung between PG and AG-effective lesions and sex between PG- and AG-effective patients. P-value less than 0.05 was considered statistically significant.max values of the control data in PG and AG were 7.92±7.80 and 7.35±6.54, respectively. There was no significant difference in the SUVmax of the control data between the PG and AG methods (P=0.09). The average SUVmax of the gated data was 8.99±7.94 in PG and 7.60±6.70 in AG. Based on the findings, the average SUVmax of PG was significantly higher than that of AG (P<0.01) (The average SUV(P<0.01) .max after gating. Similarly, 24 out of 44 (54.5%) lesions showed increased SUVmax after gating with AG. There was a statistically significant difference between the SUVmax of the control and gated data in PG (P<0.01), while there was no significant difference in AG (P=0.07)  lesions showed increased SUV(P=0.07) .max of the control data of PG and that of AG in the upper  and lower  lesions. The average SUVmax of PG was significantly higher than that of AG in both the upper  and lower  lesions.The average IR was 21.4±25.6% in PG and 4.0±14.4% in AG. As it can be seen, the average IR of PG was significantly higher than that of AG (P<0.01) . There wmax of the control data and that of the gated data in PG both in the upper (P<0.05) and lower (P<0.01) lesions. However, there was no significant difference between the SUVmax of the control and gated data in AG both in the upper (P<0.10) and lower (P<0.25) lesions.The average IR of PG was significantly higher than that of AG both in the upper  and lower  lesions . There wThere were 32 (72.7%) PG-effective and 12 (28.3%) AG-effective lesions. The average diameter of the PG- and AG-effective lesions was 24.7±17.4 and 20.3±11.3 mm, respectively. Among 32 PG-effective lesions, the number of upper-lung and lower-lung lesions was 12 and 18, respectively. The corresponding numbers in AG-effective lesions were 3 and 9, respectively. There was no significant difference in the average diameter or position of lesions between the PG- and AG-effective lesions .There were 23 (74.2%) PG-effective and 8 (25.8%) AG-effective patients. Among the PG-effective patients, there were 7 males and 16 females, while among AG-effective patients, there were 4 males and 4 females. The average age of PG- and AG-effective patients was 70.9±12.3 and 67.9±9.8 yrs, respectively. There was no significant difference in sex or age between PG- and AG-effective patients .max of lesions to compare the efficacy of PG and AG methods because it is independent at the volume of FDG avid lesion which is likely to change by using RG methods. In our study of lung adenocarcinoma lesions, the average IR of PG was higher than that of AG, which suggests that PG was more effective than AG in measuring FDG accumulation.In this study, we aimed to investigate the IR of the SUVIn addition, we evaluated the same findings in the upper and lower lesions. The analysis suggested that RG methods, especially PG, for lung adenocarcinoma lesions under free-breathing conditions should be applied independently of the position of lung lesion.Two studies have directly compared PG and AG in clinical use , 11. ElmIn the mentioned study, there was a significant difference between the two methods. Jani et al. found that AG was more effective than PG in determining the internal target volume of lung tumors ; howeverWe noted that the acquisition time of PET data should be the same to compare the tracer uptake between AG and PG methods. We divided 10 min of the acquisition data into five phases to obtain the PG data, while the AG data were determined as 25% of the total 8-min acquisition data. The total acquisition time in the two methods was consequently equivalent to that of a 2-min free-breathing scan. This is one of the reasons why the IR of the PG method was higher than that of AG in our study.Another reason is the difference in data collection algorithms. AG data contain data of multiple respiratory phases which may result in a more dispersed localization of tumor. On the other hand, RG data were obtained in a more restricted phase of the respiratory cycle. In general, AG assumes that the respiratory amplitude and the corresponding position of the lung lesion are constant in the respiratory cycle. However, they are speculated to change by cycle and might result in a more disperse location of lung lesion in AG. Overall, different amplitude ranges by the respiratory cycle may be needed for optimal gating in AG.Approximately 70% of the patients were PG-effective in this study, suggesting that PG should be used as the primary RG method for lung adenocarcinoma patients. However, there were no significant differences in sex, age, diameter of the lesion, or position of the lesion between the AG- and PG-effective patients. Therefore, it remains difficult to identify AG-effective patients before a PET study.We applied PG after AG in this study. FDG uptake is critically affected by the interval between FDG injection and data acquisition . This shWe used the best data of PG, whereas the conditions of AG were fixed. Grootjans et al. in their report suggested that a narrower bandwidth probably provides a better effect in the AG method . In our Overall, data from smaller rates  showed better IR than higher rates (data not shown). This is one of the reasons we selected a 25% rate for AG. The second reason is that we aimed to compare AG and PG data over the same accumulation time (2 min), as similar conditions are desirable.max and IR values of PG and AG methods to clarify which method is preferable for measuring FDG accumulation in lung adenocarcinoma. We found that PG was more effective for measuring FDG accumulation of lung lesions under free-breathing conditions in comparison with the AG method. Therefore, PG should be selected primarily for most lung adenocarcinoma patients.We compared the SUV